It is important to understand the mechanism(s) responsible for repairing both spontaneous and double-strand-break (DSB)-induced mitotic recombination events in eukaryotic cells. For example, in man, such events can lead to the generation of homozygous recessive oncogene that can potentiate cancer. As many of the proteins responsible for DNA damage repair in yeast and man are conserved, we propose to use yeast as an experimental system to study t s problem in both wild type and radiation sensitive cells. We will use recombination assays that we have already developed as well as new assays that we propose in this application. We will also attempt to define the biochemical activities of some of the key protein products involved in this process. Specifically, (1) we will explore alternative recombination pathway by searching for suppressor mutations that increase recombination in a rad1 rad52 double mutant since this double mutant exhibits the lowest level of spontaneous direct repeat recombination. We have thus far isolated two suppressors. One maps in RPA1, which encodes one of the three subunits of yeast Replication Factor- A. The properties of this mutation, rpa1-D228Y, will be analyzed both genetically and biochemically. The other suppressor, srr2-l, is as yet uncharacterized and we will attempt to clone the wild type copy help determine its role in suppression of rad1 rad52 double mutants. (2) We will assay both newly discovered recombination mutants singularly and in combination with various radiation sensitive mutations to determine their effects on the repair of HO endonuclease-induced double-strand-breaks. Intermediates will be investigated using the well-characterized HO- stimulated mating type interconversion assay as well as an HO-catalyzed direct repeat recombination assay that we will develop. (3) One of the central repair and recombination proteins in yeast is encoded by RAD52. We propose to examine its biochemical role by purifying the protein and developing in vitro assays to test possible functions. We will further explore the Rad52p protein-protein interaction with Rad5lp, a homologue of bacterial RecA protein, to determine the function of this complex. The studies outlined in this proposal should aid in the understanding of basic mechanisms of the repair of spontaneous and double-strand-break initiated lesions in eukaryotic cells.